The central concern of clinical chemistry is the qualitative and quantitative determination of specific analytes in samples. Of special concern is the analysis of body fluid samples, such as blood, serum, urine, and so forth. Determination of the presence and/or amount of various analytes, followed by comparison to established parameters determines diagnosis of diseased or abnormal states.
The literature on analytical determination of body fluid samples is an enormous one, as the art has investigated the determination of, e.g., glucose, cholesterol, creatine, sarcosine, urea, and other substances in samples of blood, serum, urine, and so forth.
The early clinical literature taught various non-enzymometric methods for determining analytes. Exemplary of this are the early glucose determination tests taught by Kaplan and Pesce in Clinical Chemistry: Theory, Analysis and Correlation (Mosby, 1984), pages 1032-1042. Such tests include the reduction of copper ions, reaction of copper with molybdate, and so forth. As this reference points out, these methods are insufficiently accurate, due to poor specificity, and interference by other analytes. One method described by Kaplan, et al. is the alkaline ferricyanide test. This method involves heating a solution containing glucose in the presence of ferricyanide, under alkaline conditions. The reaction: ##STR1## is accompanied by a change in color from yellow to colorless. Either this decrease in color is measured or the reaction of the colorless ferrocyanide ion with a ferric ion to form the intensely colored precipitate "Prussian Blue" is measured.
These early "chelation" type tests became replaced by more specific assays as enzymology became a more developed science. Enzymes are known for their extreme specificity, so via the use of an appropriate enzyme, the skilled artisan could determine, rather easily, whether or not a particular analyte is present, and how much. These enzymatic systems must be combined with indicator systems which, in combination with the enzyme reaction, form a detectable signal. Kaplan describes a glucose-hexokinase system, as well as a glucose oxidase system, and these are fairly well known to the art. They are used in connection with indicator systems such as the "coupled indicators" known as Trinder reagents, or oxidizable indicators such as o-tolidine and 3,3', 5,5'-tetramethylbenzidine. In such systems, reaction of the enzyme with its substrate yields a surplus of electrons carried by the enzyme, which are removed by the indicator systems. Color formation follows, indicating presence, absence, or amount of analyte in the sample.
The patent literature is replete with discussions of such systems. A by no means exhaustive selection of such patents include 4,680,259, 4,212,938, 4,144,129 and 3,925,164 (cholesterol oxidase); 4,672,029, 4,636,464, 4,490,465 and 4,418,037 (glucose oxidase); and 4,614,714 (L-glutamic acid oxidase). All of these enzymatic systems "oxidize" their substrates (i.e., the analyte in question) in that they remove electrons therefrom.
Once the analyte loses its electrons, it plays no further part in the determination reaction. As indicated, supra, the electrons may be transferred into a color forming systems, such as the Trinder system described in U.S. Pat. No. 4,291,121, or a tetrazolium system, such as is described in, e.g., U.S. Pat. No. 4,576,913. These systems employ substances known as "mediators" "electron transfer agents" or "electron shuttles" which remove the electrons from the enzymes. Eventually, the mediators release the electrons. The mediators can either absorb one, or two electrons per molecule of mediator. Ferricyanide, one preferred mediator, picks up one electron per molecule. The 4,576,913 patent, described supra, e.g., teaches another mediator, i.e., phenazine methosulfate, in combination with a tetrazolium salt. It is the latter which serves as the indicator. The use of these mediators enables one to proceed without oxygen. Normally, in a glucose determination reaction, oxygen is necessary to remove electrons from the reduced enzyme. This produces hydrogen peroxide: ##STR2## with the hydrogen peroxide taking part, in the presence of peroxidase, in reactions leading to formation of a color.
It is sometimes not desirable to use oxygen, or aerobic systems, because of various problems inherent in such systems. For example, in these reactions, the reaction is dependent on the partial pressure of O.sub.2 in the atmosphere. In addition, because the O.sub.2 must permeate throughout the entire test medium, the design of such media must be adapted to permit such permeation. There is interest, then, in indicator systems which are anaerobic, such as those where a mediator is used in connection with the indicator, or electrochemical systems using the mediator alone. There exists a need for anaerobic systems which utilize indicator reactions producing a detectable signal, such as a color.
While indicator systems of the type described supra are available, there is a difficulty with these in that the indicator molecules themselves are frequently unstable and do not have long shelf lives. There is therefore an interest in systems which utilize stable molecules which can form a detectable signal.
It will be recalled that Kaplan taught the formation of Prussian Blue in glucose determination, but dismissed it as a viable alternative because of the lack of specificity. Apart from this, the severe conditions under which the reactions are taught to take place are totally unsuitable for enzymatic assays. The reaction Kaplan teaches requires boiling the solutions. Enzymes are protein molecules, and inactivation via denaturing is characteristic of what happens when proteins are boiled. Thus, the skilled artisan, screening the heat parameters of Kaplan would avoid this teaching for enzymatic assays.
Mention of the Prussian Blue system is found in the aforementioned U.S. Pat. No. 4,576,913. This patent teaches a glycerol dehydrogenase which operates in a fashion similar to oxidases in that it teaches removal of two electrons from its substrate molecule. Column 5 of the patent refers to the Prussian Blue system (referred to as "Berlin Blue") as an the indicator.
This patent, however, must be read as a whole, and especially its teaching about the enzyme's operability. Enzymes are extremely pH sensitive, and the enzyme of the '913 patent is said to operate in a pH range from 6.0 to 10.0, and optimally at 7.0 to 8.5. The teachings, therefore, would suggest to the artisan that since the glycerol dehydrogenase operates at alkaline pHs, the adaptation of the Prussian Blue system to enzyme detection would be at alkaline pHs. However, ferric salts precipitate at alkaline pHs, which would eliminate them from participating in a reaction to form Prussian Blue under the conditions Adachi describes as necessary.
Refinements of the Prussian Blue based assay systems are described in U.S. Pat. No. 4,929,545, the disclosure of which is incorporated by reference. This patent teaches that ferrocyanide ions react with ferric ions supplied from the ferric ion containing salt Fe.sub.3 (SO.sub.4).sub.2. The system can be used for determination of various analytes, including glucose and cholesterol.
A system similar to the Prussian Blue system is one based upon the chelator ferrozine. U.S. Pat. No. 4,701,420, the disclosure of which is incorporated by reference, describes the reaction. Essentially, the system uses an electron transfer agent together with an NAD(P)H/NAD(P) system. In essence, the reaction involves the transfer of an electron to NAD(P)H from analyte, followed by transfer to the electron transfer agent. In turn, the transfer agent shuttles the electron to a ferric ion containing complex, thereby generating ferrous ions. The ferrous ions then combine with a second material, leading to formation of a colored material. A series of possible materials are described as being useful as complexing agents for the ferric ion. The materials described in the '420 patent are extremely strong chelating agents.
It has now been found, surprisingly, that chelating agents not described as useful in indicator systems and which are weaker chelating agents than these described in the art are much more useful in indicator systems. Thus, these chelating agents are an important part of the invention described herein, which is a novel composition useful for determining analytes. This invention is described in more detail in the disclosure which follows.